Rational Design of Adenylation Enzyme Inhibitors Adenylation enzymes play central roles in diverse biological pathways across all three domains of life, mediating processes such as protein translation, fatty acid and amino acid metabolism, natural product biosynthesis, and ubiquitin conjugation. These enzymes catalyze a two-step reaction involving initial ATP- dependent adenylation of a carboxylic acid substrate to form a tightly-bound acyl-AMP (acyl adenylate) intermediate, followed by attack of a nucleophile on this mixed anhydride to form an ester, thioester, or amide product. Strikingly, while all adenylation enzymes catalyze this same general reaction, at least six distinct protein folds have been identified within this mechanistic superfamily. Inhibitors of adenylation enzymes have important potential biomedical applications in infectious diseases, cancer, cardiovascular disease, metabolic disease, immunopathologies, and neurodegenerative disorders. We propose herein to continue our successful, long-term program on the rational design of adenylation enzyme inhibitors. We are advancing an inhibitor design platform that leverages mechanistic and structural information and is general for all classes of adenylation enzymes. Our previous efforts have yielded novel antibacterials targeting bacterial siderophore, glycolipid, and menaquinone biosynthesis. We have also developed semisynthetic protein inhibitors of ubiquitin/ubiquitin-like modifier E1 activating enzymes that have provided profound mechanistic insights into the functions of these enzymes. Our goals for the next project period are to develop selective inhibitors of non-ribosomal peptide biosynthesis using a macrocyclic design, to develop optimized menaquinone biosynthesis inhibitors as new antibacterials, and to develop protein-based and small-molecule inhibitors of E1 activating enzymes to probe their molecular mechanisms and biological functions in cancer. Systematic correlation of physicochemical properties with bacterial uptake will also be studied. This work will have broad impacts in structure-based design, antibacterial medicinal chemistry, and enzymology, and will be carried out through established multidisciplinary collaborations comprising combined expertise in organic synthesis, medicinal chemistry, pharmacology, biochemistry, microbiology, and structural biology.